The pairing of homologous chromosomes at meiosis I is an essential step in the production of euploid gametes. Failures in pairing lead to chromosome missegregation events (nondisjunction), a leading cause of human genetic syndromes. A novel pairing assay has been developed in the male fruit fly, Drosophila melanogaster, that allows one to assay for the ability of selected sequences to facilitate sex chromosome meiotic pairing (i.e. act as pairing sites). The assay employs an X chromosome that is deleted for the endogenous meiotic pairing sites and a partner Y chromosome into which X chromosome euchromatin has been inserted. Genetic tests of sex chromosome transmission indicate that these ?synthetic? pairing sites are capable of all steps required for proper XY segregation, including homolog recognition, adhesion, orientation and disjunction. Molecularly defined CRISPR-mediated deletions of putative pairing sites will be created to identify sequence, position and/or size-specific requirements for pairing sites. Confocal microscopy will be used to examine in situ hybridization of probes to putative pairing sites, and in combination with genetic tests of chromosome transmission, will determine if there are different requirements for pairing versus homolog adhesion versus disjunction. Mutations in genes encoding the putative adhesion complex proteins Tef, MNM and SMN will be tested to define the roles of these genes in effecting adhesion between X euchromatin sequences. As male fly lack recombination, these studies may reveal insights into potentially conserved aspects of homology recognition and pairing that are independent of recombination-associated processes.